PROJECT SUMMARY The main route for HIV acquisition in women of all ages is sexual transmission. While younger women (15?24 years) are at high risk for HIV infection in endemic areas, an increasing incidence of new HIV infections in older women (age >50 years) is observed worldwide. Yet, the mucosal events that lead to the prevention or acquisition of HIV and how mucosal protection is affected with aging are largely unknown. Thus, it is critical to identify the early mucosal mechanisms that prevent HIV infection of target cells and how this protection is modified with aging, in order to develop effective preventive approaches for younger and older women. Neutrophils are abundant throughout the female reproductive tract (FRT) in pre- and post-menopausal women and are distinct from neutrophils present in blood. However, despite their key location at mucosal surfaces in the FRT (the main site for HIV exposure in women) and their critical role in innate immunity, the extent to which FRT neutrophils contribute to protection against HIV infection in women is unknown. The PI's research group recently discovered that, neutrophils from the human FRT undergo NETosis in response to HIV, which prevents infection of CD4+ T cells. NETosis is a phenomenon characterized by the release of Neutrophil Extracellular Traps (or NETs), which are segments of DNA associated with granular proteins with antimicrobial activity. The research team further found that the magnitude of HIV-induced NETosis is significantly reduced in post-menopausal women. Based on these preliminary results, the hypothesis being tested here is that HIV-induced NETosis of neutrophils represents a previously unrecognized level of mucosal innate protection against HIV in the FRT that is compromised with aging. This hypothesis will be tested through three specific aims. First, experimental studies will be performed to determine how FRT NETs prevent HIV infection (Aim 1); then, to identify the mechanisms by which HIV triggers NETosis of FRT neutrophils (Aim 2); and, finally, to elucidate why HIV-induced NETosis is impaired in post-menopausal women (Aim 3). The proposed studies will investigate purified neutrophils isolated from different sites in the FRT (endometrium, endocervix, and ectocervix). NETosis will be assessed with a quantitative, high-throughput, time-lapse imaging approach. The overall objective will be to determine how NETs contribute to anti-HIV mucosal protection in the FRT. It is expected that these studies will result in the identification of NETosis as a previously unrecognized form of immune protection against HIV in the FRT, one that has the potential of leading to a paradigm shift in our understanding of HIV acquisition. The identification of the mechanisms triggering NETosis in response to HIV, and how these mechanisms are compromised in older women, will have significant translational impact and serve as the foundation for novel prevention approaches for women of all ages.